The bioactive lipid mediator sphingosine 1-phosphate (S1P) is considered to be involved in the development of insulin resistance (IR) via effects on oxidative stress; the mechanism however is not yet fully revealed. To this end, we investigated the role and mechanism of S1P on hepatic IR. We found that treatment of the normal human liver cell LO2 with 1000 nM insulin for 48 h reduced glucose uptake and increased serine phosphorylation of insulin receptor substrate-1, indicating a reduction in insulin receptor signaling. Moreover, the same concentration of insulin caused accumulation of reactive oxygen species (ROS) in the cytosol and mitochondria, and enhanced expression of the antioxidant transcription factor (Nrf2) and upregulated Nrf2 nuclear translocation. Using known inhibitors and donors of ROS (H2O2, ·O2-, ·OH), the results demonstrated the differential roles for the specific ROS in regulating IR in LO2 cells, with H2O2 having a more significant inhibitory role compared with ·O2- and ·OH. Cell treatment with S1P at 0.1-5.0 μM reversed the effects of high insulin concentrations on ROS generation, glucose uptake, and insulin signaling. H2O2 also reversed the beneficial effects of S1P in alleviating IR. These results show that H2O2 signaling plays a key determinant in hepatic IR induced by insulin. S1P can ameliorate hepatic IR by reducing mitochondrial ROS generation, and the possible anti-IR effect mechanism may be involved in H2O2 signaling. Copyright © 2019. Published by Elsevier B.V.